New study shows normal-looking clones may be abnormal

Scientists have found the first evidence to show that even seemingly normal-looking clones may harbor serious abnormalities affecting gene expression that may not manifest themselves as outward characteristics. The findings, reported in the July 6 issue of Science by researchers at the Whitehead Institute for Biomedical Research and University of Hawaii, confirm the previous suspicion that reproductive cloning is not only inefficient, but may actually be unsafe.

In the study, scientists from Rudolf Jaenisch's laboratory and Ryuzo Yanagimachi's laboratory sought to examine the mechanisms underlying poor survival and gross overgrowth in cloned animals. They made mouse clones from embryonic stem (ES) cells and monitored the activity of imprinted genes--developmental genes that are controlled by special tags that do not affect the base sequence itself. They looked to see if these tags were faithfully reproduced in cloned mice and in the donor cells used to make the clones. This would tell them if the problems with cloning resulted from aberrations in donor cells or as a result of the cloning procedure itself.

Scientists found to their surprise that much of the problem lay in the make up of the donor ES cells, which they found to be extremely unstable in culture. As they divide in culture, these cells lose the tags that tell an imprinted gene to be either turned on or off during development. The researchers found that even clones made from sister stem cells had differences in their gene expression. Despite this instability, many embryos survived to adulthood, suggesting that mammalian development is surprisingly tolerant to aberrant gene regulation. "This suggests that even apparently normal clones may have subtle aberrations of gene expression that are not easily detected in the cloned animal," says Jaenisch.

The cloning procedure involves removing the nucleus, or the genetic command center, of an egg and replacing it with the nucleus from an adult cell or an ES cell. Ideally, the egg resets the developmental clock of the nucleus back to a state compatible with early embryonic growth and gives rise to a new organism that is genetically identical to the donor cell. Researchers had thought that using the nucleus from ES cell might require less reprogramming than using the nucleus from an adult cell, such as a mammary cell, which has already committed to a certain function.

ES cells are unique in that they can give rise to an entire organism without using the nuclear transfer procedure. The researchers found that animals developed from mouse embryonic stem cells by this technique exhibited irregular gene expression just as animals derived from nuclear transfer using embryonic stem cells confirming that these abnormalities likely existed in the cells before the cloning procedure was performed.

"While embryonic stem cells may be easier to developmentally reprogram than adult cells, they may have a different set of problems arising from errors occurring during culturing that cannot be fixed by cloning," says David Humpherys, a senior graduate student in the Jaenisch lab.

This may mean researchers will need to find ways to better preserve gene regulatory tags during cell culture techniques. Researchers also still need to test whether animals cloned from adult cells will show similar defects.

"However, despite their instability, it is important to remember that embryonic stem cells when combined with normal cells--as in making chimeras or when used in transplantation--may function fine. In other words, ES cells might work fine when used as cell therapy, but when they are used to make whole animals would likely produce organisms that are abnormal," says Jaenisch.

"While these new results are a concern for reproductive cloning, misexpression of imprinted genes likely has little significance for therapeutic cloning. Imprinted genes have defined roles in fetal development, but may serve no crucial role in the differentiated cells of the adult," says Humpherys.

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